This invention generally relates to methods and compositions for modulating cell adhesion and for inhibiting the interaction between integrins and their ligands. In particular, the invention relates to peptides that selectively inhibit xcex14 integrins.
A. Integrins
Integrins are a family of cell surface proteins that mediate adhesion between cells (cell-cell adhesion) and between cells and extracellular matrix proteins (cell-ECM adhesion). Integrins are heterodimeric structures composed of noncovalently bound xcex1 and xcex2 subunits. In humans there are at least 15 different xcex1 and eight different xcex2 subunits, and these can combine to form proteins with diverse biological activities and ligand specificities.
The integrins play important roles in many diverse biological processes including platelet aggregation, tissue repair, angiogenesis, bone destruction, tumor invasion, inflammation, and immune reactions. The integrins are, therefore, important targets for therapeutic intervention in human disease.
Integrin xcex1IIbxcex23 (glycoprotein IIb/IIIa complex) binds fibrinogen on the platelet surface and mediates platelet aggregation. Integrin xcex1vxcex23 is predominantly expressed on endothelial cells and plays an important role in angiogenesis. Integrin xcex1vxcex23 is also expressed on osteoclasts and participates in bone destruction. Integrin xcex15xcex21 is widely distributed on a variety of cells; it plays a critical role in cell adhesion to extracellular matrix as well as in the formation of tissues and organs during embryonic development. All three integrins xcex1IIbxcex23, xcex1vxcex23 and xcex15xcex21 recognize RGD sequences in the adhesive ligands.
One example of the therapeutic targeting of integrins is the use of integrin inhibitors as antithrombotic agents. Peptides and peptidomimetics that block the adhesion of GPIIb/IIIa to fibrinogen can prolong bleeding times and prevent thrombotic occlusion in vivo. One group of naturally occurring peptides that inhibit platelet aggregation by interfering with fibrinogen binding to GPIIb/IIIa has been called the xe2x80x9cdisintegrins.xe2x80x9d
B. Disintegrins
The disintegrins are a family of low molecular weight cysteine-rich peptides that have been isolated from the venom of various snakes (reviewed in Niewiarowski et al., Seminars in Hematology 31(4):289-300 (1994)). Most disintegrins described to date contain an RGD motif. RGD is a recognition site for many integrins, and disintegrins inhibit fibrinogen binding to GPIIb/IIIa, as well as the binding of other ligands to RGD-dependant integrins on the surface of cells. Peptides modeled on the structure of disintegrins have potential clinical applications in the prevention and treatment of coronary thrombosis, stroke, and other vascular diseases.
The first disintegrin described in the literature, trigramin, was identified and characterized on the basis of its ability to block platelet aggregation and inhibit fibrinogen binding to xcex1IIbxcex23. Trigramin contains 72 amino acids including 12 cysteines, is linked by S-S bonds, and contains an RGD sequence. Subsequently several other RGD containing viper venom disintegrins of similar size were isolated.
A 49 amino acid disintegrin, called echistatin, has been isolated from the venom of Echis carinatus (Gan et al., J. Biol. Chem. 263:19827-32 (1988)). Like other disintegrins, echistatin contains an RGD sequence and inhibits GPIIb/IIIa binding of fibrinogen. Echistatin has been called a xe2x80x9cpromiscuous disintegrinxe2x80x9d because it blocks xcex1IIbxcex23, xcex1vxcex23 and xcex15xcex21 with similar potency.
NMR studies on RGD-containing disintegrins shows that the RGD sequence is located in a mobile loop joining two strands of xcex2 sheet protruding from the protein core (reviewed in Niewiarowski et al., Semin. Hematol. 31:289-300 (1994)). The disulfide bonds around the RGD sequence maintain the hair-pin loop conformation in each peptide, and this conformation seems to be important for potency and selectivity.
The disintegrin eristostatin, originally described as a potent inhibitor of xcex1IIbxcex23, also inhibits human melanoma cell (MV3) metastases in immune deficient mice. It has been suggested that this effect is mediated by altering the function of an xcex14 integrin expressed on MV3 cells (Danen et al, Exp. Cell Res. 238:188-196 (1998)).
C. The xcex14 Integrins
The xcex14 integrins, xcex14xcex21 and xcex14xcex27, are expressed on leukocytes and lymphoid cells, and play a major role in inflammation and auto-immune diseases.
The xcex14xcex21 integrin (which has also been called VLA4, very late activation antigen 4) mediates cell adhesion to vascular cell adhesion molecule-1 (VCAM-1), an adhesive molecule belonging to the IgG superfamily which is expressed on endothelial cells at sites of inflammation. The integrin xcex14xcex21 also binds to alternatively spliced variants of fibronectin containing connecting segment 1 (CS-1).
The xcex14xcex27 integrin binds to the gut homing receptor mucosa addressin cell adhesion molecule-1 (MadCAM-1) and to a lesser extent to CS-1 and VCAM-1.
Cytokine activated leukocytes express xcex14xcex21 and xcex14xcex27 integrins. Interaction of these integrins with VCAM-1 or MadCAM-1 (which are also up-regulated by cytokines) on endothelium mediates capillary infiltration by leukocytes, which can lead to tissue and organ destruction. Selectins and xcex22 integrins also contribute to this process. Altevost et al. reported that the xcex14 subunit itself is a ligand for xcex14xcex21 and xcex14xcex27 integrins (J. Exp. Med. 182:345-55 (1995)), suggesting that xcex14 integrins may play a role in leukocyte communication during the immune response.
Activation and up-regulation of xcex14xcex21 or xcex14xcex27 on lymphocytes or macrophages is believed to play a significant role in the progression of many disease states, including insulin dependent diabetes mellitus, multiple sclerosis, rheumatoid arthritis, ulcerative colitis, arteriosclerosis, asthma, allergy, organ rejection, and restenosis of arteries after surgery or angioplasty. The xcex14 integrins are therefore targets for therapeutic intervention in a variety of inflammatory, auto-immune, and other diseases.
D. The xcex14 Integrins as Therapeutic Targets
There are several animal models of inflammatory and autoimmune diseases in which endothelial infiltration by lymphocytes and organ destruction are blocked by anti-xcex14 monoclonal antibodies. As an example, anti-xcex14 antibody inhibits lymphocyte infiltration of Langerhans islets in NOD mice, thus preventing development of spontaneous insulin dependent diabetes (Yang et al., Proc. Natl. Acad. Sci. USA 4 91:12604-08 (1994)). Anti-xcex14 monoclonal antibodies have also shown in vivo efficacy in animal models of asthma (Abraham et at., J. Clin. Invest. 93:776 (1994)), multiple sclerosis (Yednock et al., Nature 356:63 (1992)), inflammatory bowel disease (Podoisky et al., J. Clin. Invest. 92:372 (1993)), contact hypersensitivity (Chisholm et al., Eur. J. Immunol. 23:682 (1993)), and cardiac allograft rejection (Isobe et al., J. Immunol. 153:5810 (1994)).
There have been several attempts to isolate naturally occurring or to develop synthetic inhibitors of xcex14xcex27 and xcex14xcex21. Synthetic inhibitors that have been reported include cyclic RGD peptides and short peptides based on the sequences of MadCAM-1, VCAM-1, and CS-1. These peptides are typically active in vitro at the micromolar level.
Molossi et al. (J. Clin. Invest. 95:2601-10 (June 1995)) reported that blockade of xcex14xcex21 (VLA-4) integrin binding to fibronectin with CS-1 peptide reduces accelerated coronary arteriopathy in rabbit cardiac allografts. The sequence of the CS-1 peptide was phenylacetic acid-Leu-Asp-Phe-d-Pro-amide.
Kogan et al. (WO 96100581) reported that cyclic peptides modeled after a portion of the CS-1 peptide inhibited the binding of xcex14xcex21 integrin to VCAM-1 at concentrations of peptide less than about 10 xcexcM.
Kogan et al. (U.S. Pat. No. 5,510,332) reported that a peptide comprising the LDV domain of CS-1 peptide inhibited the binding of xcex14xcex21 integrin to VCAM-1 with an IC50 of 30 xcexcM.
Shroff et al. (Bioorganic and Medicinal Chemistry Letters 6(21):2495-2500 (1996)) reported a series of peptides based on the N-terminal domain of MAdCAM-1 inhibited the binding of HUT78 cells activated with Mn++ to MAdCAM-1 with an IC50 of 5 to  greater than 1000 xcexcM. Cyclic peptides based on an N-terminal conserved motif in VCAM-1 have also been reported to inhibit VCAM/xcex14xcex21 mediated leukocyte adhesion (Wang et al., Proc. Natl. Acad. Sci. USA 92:5714 (1995)).
Cyclic RGDS (SEQ ID NO:15) peptides have been reported as strong inhibitors of xcex14xcex21 and xcex14xcex27 integrins (Cardarelli et al., J. Biol. Chem. 269:18668-73 (1994); Yang et al. Eur. J. Immunol. 28:995-1004 (1998)).
Vanderslice et al. (J. Immunol. 158:1710-18 (1997)) recently reported that a cyclic peptide based on the LDV sequence of CS-1 inhibited xcex14xcex21 dependent binding of lymphocytes to VCAM and CS-1 with an IC50 of 1-3 xcexcM.
There is a need for potent and specific inhibitors of xcex14 integrins, and for methods of specifically inhibiting the binding of xcex14 integrins to cellular ligands.
The present invention relates to the discovery of the EC-3 protein, a heterodimeric disintegrin that is an extremely potent antagonist of xcex14 integrins. EC-3 proteins and peptides inhibit adhesion of cells expressing xcex14 integrins in an RGD-independent manner.
The invention provides a substantially purified EC-3 protein, isolated from E. carinatus venom and characterized by: (a) an apparent molecular weight of about 14,762 Da, as determined by electrospray ionization mass spectrometry; (b) elution from a C-18 HPLC column at about 40% acetonitrile; and (c) the ability to inhibit adhesion of Jurkat cells to VCAM-1.
The invention also provides a substantially purified EC-3A peptide, isolated from EC-3 protein which has been reduced and alkylated, and characterized by: (a) a molecular mass of about 8478 Da in its ethylpyridylated form, as determined by electrospray ionization mass spectrometry; (b) elution from a C-18 HPLC column at about 42% acetonitrile; and (c) the ability to inhibit adhesion of K562 cells to fibronectin.
The invention further provides a substantially purified EC-3B peptide, isolated from EC-3 protein which has been reduced and alkylated with vinylpyridine, and characterized by: (a) a molecular mass of about 7950 Da in its carboxymethylated form, as determined by electrospray ionization mass spectrometry; (b) elution from a C-18 HPLC column at about 46% acetonitrile; and (c) the ability to inhibit adhesion of Jurkat cells to VCAM-1.
One preferred embodiment of the invention is a substantially purified EC-3A peptide comprising the sequence SEQ ID NO:19, or a biologically active fragment or derivative thereof. A more preferred embodiment is a substantially purified peptide comprising the sequence SEQ ID NO:2, or a biologically active fragment or derivative thereof.
Another preferred embodiment of the invention is a substantially purified EC-3B peptide comprising the sequence SEQ ID NO:20, or a biologically active fragment or derivative thereof. A more preferred embodiment is a substantially purified peptide comprising the sequence SEQ ID NO:3, or a biologically active fragment or derivative thereof.
A further embodiment of the invention is a substantially purified EC-3 protein comprising two subunits, wherein one subunit comprises the sequence SEQ ID NO:19 or a biologically active fragment or derivative thereof and one subunit comprises the sequence SEQ ID NO:20 or a biologically active fragment or derivative thereof.
The invention is also directed to a biologically active peptide fragment of EC-3B having the sequence X-Y-Met-Leu-Asp-Z, where X is H or a blocking group, Y is zero or more amino acids, and Z is OH or zero or more amino acids. In some preferred embodiments the biologically active peptide is from about 3 to about 20 amino acids. In a more preferred embodiment the peptide has the sequence SEQ ID NO:16. In another more preferred embodiment the peptide has the sequence SEQ ID NO:14.
Another aspect of the invention is a substantially purified nucleic acid encoding a protein or peptide according to the invention. One embodiment of the invention is a vector comprising a nucleic acid encoding a protein or peptide according to the invention. Another embodiment of the invention is a recombinant cell comprising a nucleic acid encoding a protein or peptide according to the invention.
The invention further provides an antibody which specifically binds to a protein or peptide according to the invention. The antibody may be a monoclonal antibody or a polyclonal antibody or an antibody fragment that is capable of binding antigen. One aspect of the invention is a hybridoma that produces a monoclonal antibody which specifically binds to a protein or peptide according to the invention.
Another aspect of the invention is a substantially purified echistatin polypeptide in which the Arg-Gly-Asp residues at positions 24-26 are replaced by Met-Leu-Asp, or a biologically active fragment or derivative thereof.
The invention further provides a method of isolating a peptide that binds to an integrin of interest from venom comprising: (a) dissolving venom in a solvent, (b) centrifuging the dissolved venom to remove high molecular weight proteins, (c) fractionating the supernatant from step (b), (d) immobilizing the fractions from step (c) on a solid support, (e) adding detectably labeled cells which express the integrin of interest to the immobilized fractions, (f) detecting the number of cells bound to each immobilized fraction, and (g) isolating peptide from those fractions which showed enhanced cell binding in step (f).
Another aspect of the invention is a composition comprising a pharmaceutically acceptable carrier and a protein, peptide, or nucleic acid according to the invention.
The invention encompasses a method of inhibiting the binding of an xcex14 integrin to VCAM-1 comprising contacting a cell that expresses the xcex14 integrin with an effective amount of a protein or peptide according to the invention. In preferred embodiments the integrin is xcex14xcex21 or xcex14xcex27.
The invention also encompasses a method of inhibiting the binding of xcex14xcex27 integrin to MadCAM-1 comprising contacting a cell that expresses xcex14xcex27 with an effective amount of a protein or peptide according to the invention.
The invention further encompasses a method of inhibiting the binding of an xcex14 integrin to CS-1 comprising contacting a cell that expresses the xcex14 integrin with an effective amount of a protein or peptide according to the invention.
One preferred embodiment of the invention is a method of inhibiting the interaction between cells expressing an xcex14 integrin and VCAM-1 in a patient in need of such treatment comprising administration of a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein or peptide according to the invention.
Another preferred embodiment of the invention is a method of inhibiting the interaction between cells expressing an xcex14 integrin and MadCAM-1 in a patient in need of such treatment comprising administration of a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein or peptide according to the invention.
A further preferred embodiment of the invention is a method of, inhibiting the interaction between cells expressing an xcex14 integrin and CS-1 in a patient in need of such treatment comprising administration of a therapeutically effective amount of a composition comprising a pharmaceutically acceptable carrier and a protein or peptide according to the invention.
The invention is also directed to the use of a protein, peptide, or nucleic acid according to the invention for the preparation of a medicament for inhibiting the interaction between cells expressing an xcex14 integrin and its ligands.
Other aspects and advantages of the present invention are described in the drawings and in the following detailed description of the preferred embodiments thereof.